Hybridization Sequencing Research Articles

Mechanical, thermal, and water absorption behaviour of jute/carbon reinforced hybrid composites

The demand for fiber-reinforced composite materials is increasing in structural applications due to their crucial characteristics such as stiffness, strength, and durability and processing benefits at low cost. In this study, jute/carbon hybrid composite laminates were investigated for the effect of fabric hybridization and stacking sequence on tensile, impact, microhardness, water absorption, and thermal behavior of the material. The hand layup process was used to fabricate the composite laminates with four different stacking sequences. The X-ray diffraction (XRD), Fourier-Transform Infrared spectroscopy (FT-IR), Thermogravimetric analysis (TGA), and Scanning Electron Microscope (SEM) were used to characterize the structural morphology and thermal stability of the fabricated composites. The experimental results exposed that the hybridization process enhanced the properties of jute reinforced composites. FT-IR and XRD analysis revealed that the alkalization process removed the binding constituents like lignin and hemicelluloses from raw jute fiber, which resulted in a higher crystallinity index. The TGA analysis proved that the hybrid composites are thermally stable at a higher temperature. The hybrid composite with Jute/Carbon/Carbon/Jute stacking patterns has the highest tensile strength of 234.68 MPa compred to other stacking sequences. The hybrid composite with Carbon/Jute/Jute/Carbon fabric stacking sequence exhibited enhanced impact strength of 108.45 kJ/m2 and better moisture resistance. The incorporation of jute with carbon declined the tensile strength and impact strength by 22% and 14%, respectively, compared to carbon-reinforced composites. The surface micrographs of the fractured samples exhibit the interfacial bonding of fiber/matrix, matrix crack, fiber fracture, and fiber pullouts.

\xc5ngstr\xf6m- and Nano-scale Pore-Based Nucleic Acid Sequencing of Current and Emergent Pathogens

State-of-the-art nanopore sequencing enables rapid and real-time identification of novel pathogens, which has wide application in various research areas and is an emerging diagnostic tool for infectious diseases including COVID-19. Nanopore translocation enables de novo sequencing with long reads (> 10 kb) of novel genomes, which has advantages over existing short-read sequencing technologies. Biological nanopore sequencing has already achieved success as a technology platform but it is sensitive to empirical factors such as pH and temperature. Alternatively, ångström- and nano-scale solid-state nanopores, especially those based on two-dimensional (2D) membranes, are promising next-generation technologies as they can surpass biological nanopores in the variety of membrane materials, ease of defining pore morphology, higher nucleotide detection sensitivity, and facilitation of novel and hybrid sequencing modalities. Since the discovery of graphene, atomically-thin 2D materials have shown immense potential for the fabrication of nanopores with well-defined geometry, rendering them viable candidates for nanopore sequencing membranes. Here, we review recent progress and future development trends of 2D materials and their ångström- and nano-scale pore-based nucleic acid (NA) sequencing including fabrication techniques and current and emerging sequencing modalities. In addition, we discuss the current challenges of translocation-based nanopore sequencing and provide an outlook on promising future research directions.

Three-dimensional chromosome conformation capture and its derived technologies

Linear chromatin is compacted into eukaryotic nucleus through a complex and multi-layered architecture. Consequently, chromatin conformation in a local or long-distance manner is strongly correlated with gene expression. Chromosome conformation capture (3C) technology, together with its variants like 4C/5C/Hi-C, has been well developed to study chromatin looping and whole genome structure. In this review, we introduce new technologies including chromosome capture combined with immunoprecipitation, nuclei acid-based hybridization, single cell and genome sequencing, as well as their application.

Heterozygous de novo variants in CSNK1G1 are associated with syndromic developmental delay and autism spectrum disorder.

The gamma-1 isoform of casein kinase 1, the protein encoded by CSNK1G1, is involved in the growth and morphogenesis of cells. This protein is expressed ubiquitously among many tissue types, including the brain, where it regulates the phosphorylation of N-methyl-D-aspartate receptors and plays a role in synaptic transmission. One prior individual with a de novo variant in CSNK1G presenting with severe developmental delay and early-onset epilepsy has been reported. Here we report an updated clinical history of this previously published case, as well as four additional individuals with de novo variants in CSNK1G1 identified via microarray-based comparative genomic hybridization, exome, or genome sequencing. All individuals (n = 5) had developmental delay. At least three individuals had diagnoses of autism spectrum disorder. All participants were noted to have dysmorphic facial features, although the reported findings varied widely and therefore may not clearly be recognizable. None of the participants had additional major malformations. Taken together, our data suggest that CSNK1G1 may be a cause of syndromic developmental delay and possibly autism spectrum disorder.

Efficient Sequencing, Assembly, and Annotation of Human KIR Haplotypes.

The homology, recombination, variation, and repetitive elements in the natural killer-cell immunoglobulin-like receptor (KIR) region has made full haplotype DNA interpretation impossible in a high-throughput workflow. Here, we present a new approach using long-read sequencing to efficiently capture, sequence, and assemble diploid human KIR haplotypes. Probes were designed to capture KIR fragments efficiently by leveraging the repeating homology of the region. IDT xGen® Lockdown probes were used to capture 2–8 kb of sheared DNA fragments followed by sequencing on a PacBio Sequel. The sequences were error corrected, binned, and then assembled using the Canu assembler. The location of genes and their exon/intron boundaries are included in the workflow. The assembly and annotation was evaluated on 16 individuals (8 African American and 8 Europeans) from whom ground truth was known via long-range sequencing with fosmid library preparation. Using only 18 capture probes, the results show that the assemblies cover 97% of the GenBank reference, are 99.97% concordant, and it takes only 1.8 haplotigs to cover 75% of the reference. We also report the first assembly of diploid KIR haplotypes from long-read WGS. Our targeted hybridization probe capture and sequencing approach is the first of its kind to fully sequence and phase all diploid human KIR haplotypes, and it is efficient enough for population-scale studies and clinical use. The open and free software is available at https://github.com/droeatumn/kass and supported by a environment at https://hub.docker.com/repository/docker/droeatumn/kass.

Effects of Chemical Treatment, Hybridization, and Hybrid Fiber Stacking Sequence and Orientation on Tensile and Impact Strength of Continuous Sisal Fiber Reinforced Polyester Composites

ABSTRACT This paper has presented the enhancement of tensile and impact strength of continuously aligned sisal fiber reinforced polyester composites through chemical treatment and hybridization. Sodium hydroxide (NaOH) and baking soda (NaHCO3) were used for sisal fiber treatment. Comparisons on the effect of sodium hydroxide and baking soda treatments on tensile and impact strength of the composites were performed. Unidirectional glass and carbon fibers were selected as hybrid fibers. Nineteen laminates were prepared as per the standards. The laminates were made of pure sisal fiber and hybridized with glass or carbon fibers at different stacking sequence, orientation, and volume fraction. The effects of glass and carbon fibers hybridization on tensile and impact strength of the composites were studied and compared. Further enhancements on the tensile and impact strength of the composites were investigated through alterations on the stacking sequence, orientation, and volume fraction of the hybrid fibers. Symmetric hybrid composites which avoided sisal/sisal plies interface have shown more than 95% and 130% rise in their tensile strength by adding about 8% volume of glass and carbon fibers respectively. Moreover, with only about 8% carbon fiber inclusion in the laminates, almost 60% improvements in the impact strength were discovered.

A systematic review on bacterial community changes after periodontal therapy with and without systemic antibiotics: An analysis with a wider lens.

This systematic review aimed to provide a comprehensive view on microbial community shifts after periodontal therapy with and without systemic antibiotics, conducted in randomized controlled trials (RCTs). Search functions in PubMed, Scopus, the Web of Knowledge, and the Cochrane Oral Health Library databases were used to locate studies published up to December 2018 that reported at least two bacteria before and after periodontal therapy. Gray literature and manual searching were done. Information about reported bacteria in those studies were extracted, and a descriptive microbial community analysis was conducted to observe trends and influencing factors on microbial dynamics. Methodological aspects were examined, including the bacterial detection method, heterogeneity of procedures, and risk of bias (RoB) of the studies. The 30 included studies reported 130 different bacterial genera. Four different detection methods were reported: cultivation, polymerase chain reaction, DNA-DNA-checkerboard hybridization, and 16S rDNA amplicon sequencing. No general compositional change between the antibiotic and placebo groups could be found after therapy on the community level. Fifty-five bacteria were reported in two or more studies. Of those, 24 genera decreased and 13 increased more frequently after antibiotic use. Great heterogeneity between procedures and variability in RoB were found among the studies. Microbial shifts occurred regardless of the use of antibiotics. Antibiotic therapy seems to induce more changes in single bacteria. The heterogeneity in methods and reporting of the included studies preclude clinical recommendations on the use or not of adjunctive antibiotics. The present results may guide further research on the topic.

Determination of a novel parvovirus pathogen associated with massive mortality in adult tilapia.

Tilapia is one of the most important economic and fastest-growing species in aquaculture worldwide. In 2015, an epidemic associated with severe mortality occurred in adult tilapia in Hubei, China. The causative pathogen was identified as Tilapia parvovirus (TiPV) by virus isolation, electron microscopy, experimental challenge, In situ hybridization (ISH), indirect immunofluorescence (IFA), and viral gene sequencing. Electron microscopy revealed large numbers of parvovirus particles in the organs of diseased fish, including kidney, spleen, liver, heart, brain, gill, intestine, etc. The virions were spherical in shape, non-enveloped and approximately 30nm in diameter. The TiPV was isolated and propagated in tilapia brain cells (TiB) and induced a typical cytopathic effect (CPE) after 3 days post-infection (dpi). This virus was used to experimentally infect adult tilapia and clinical disease symptoms similar to those observed naturally were replicated. Additionally, the results of ISH and IFA showed positive signals in kidney and spleen tissues from TiPV-infected fish. To identify TiPV-specific sequences, the near complete genome of TiPV was obtained and determined to be 4269 bp in size. Phylogenetic analysis of the NS1 sequence revealed that TiPV is a novel parvovirus, forms a separate branch in proposed genus Chapparvovirus of Parvoviridae. Results presented here confirm that TiPV is a novel parvovirus pathogen that can cause massive mortality in adult tilapia. This provides a basis for the further studies to define the epidemiology, pathology, diagnosis, prevention and treatment of this emerging viral disease.

Alternative Splicing Enhances the Transcriptome Complexity of Liriodendron chinense.

Alternative splicing (AS) plays pivotal roles in regulating plant growth and development, flowering, biological rhythms, signal transduction, and stress responses. However, no studies on AS have been performed in Liriodendron chinense, a deciduous tree species that has high economic and ecological value. In this study, we used multiple tools and algorithms to analyze transcriptome data derived from seven tissues via hybrid sequencing. Although only 17.56% (8,503/48,408) of genes in L. chinense were alternatively spliced, these AS genes occurred in 37,844 AS events. Among these events, intron retention was the most frequent AS event, producing 1,656 PTC-containing and 3,310 non-PTC-containing transcripts. Moreover, 183 long noncoding RNAs (lncRNAs) also underwent AS events. Furthermore, weighted gene coexpression network analysis (WGCNA) revealed that there were great differences in the activities of transcription and post-transcriptional regulation between pistils and leaves, and AS had an impact on many physiological and biochemical processes in L. chinense, such as photosynthesis, sphingolipid metabolism, fatty acid biosynthesis and metabolism. Moreover, our analysis showed that the features of genes may affect AS, as AS genes and non-AS genes had differences in the exon/intron length, transcript length, and number of exons/introns. In addition, the structure of AS genes may impact the frequencies and types of AS because AS genes with more exons or introns tended to exhibit more AS events, and shorter introns tended to be retained, whereas shorter exons tended to be skipped. Furthermore, eight AS genes were verified, and the results were consistent with our analysis. Overall, this study reveals that AS and gene interaction are mutual—on one hand, AS can affect gene expression and translation, while on the other hand, the structural characteristics of the gene can also affect AS. This work is the first to comprehensively report on AS in L. chinense, and it can provide a reference for further research on AS in L. chinense.

LncMirNet: Predicting LncRNA-miRNA Interaction Based on Deep Learning of Ribonucleic Acid Sequences.

Long non-coding RNA (LncRNA) and microRNA (miRNA) are both non-coding RNAs that play significant regulatory roles in many life processes. There is cumulating evidence showing that the interaction patterns between lncRNAs and miRNAs are highly related to cancer development, gene regulation, cellular metabolic process, etc. Contemporaneously, with the rapid development of RNA sequence technology, numerous novel lncRNAs and miRNAs have been found, which might help to explore novel regulated patterns. However, the increasing unknown interactions between lncRNAs and miRNAs may hinder finding the novel regulated pattern, and wet experiments to identify the potential interaction are costly and time-consuming. Furthermore, few computational tools are available for predicting lncRNA–miRNA interaction based on a sequential level. In this paper, we propose a hybrid sequence feature-based model, LncMirNet (lncRNA–miRNA interactions network), to predict lncRNA–miRNA interactions via deep convolutional neural networks (CNN). First, four categories of sequence-based features are introduced to encode lncRNA/miRNA sequences including k-mer (k = 1, 2, 3, 4), composition transition distribution (CTD), doc2vec, and graph embedding features. Then, to fit the CNN learning pattern, a histogram-dd method is incorporated to fuse multiple types of features into a matrix. Finally, LncMirNet attained excellent performance in comparison with six other state-of-the-art methods on a real dataset collected from lncRNASNP2 via five-fold cross validation. LncMirNet increased accuracy and area under curve (AUC) by more than 3%, respectively, over that of the other tools, and improved the Matthews correlation coefficient (MCC) by more than 6%. These results show that LncMirNet can obtain high confidence in predicting potential interactions between lncRNAs and miRNAs.

Clinicopathological findings of pediatric NTRK fusion mesenchymal tumors

BackgroundWhile ETV6- NTRK3 fusion is common in infantile fibrosarcoma, NTRK1/3 fusion in pediatric tumors is scarce and, consequently, not well known. Herein, we evaluated for the presence of NTRK1/3 fusion in pediatric mesenchymal tumors, clinicopathologically and immunophenotypically.MethodsWe reviewed nine NTRK fusion-positive pediatric sarcomas confirmed by fluorescence in situ hybridization and/or next-generation sequencing from Seoul National University Hospital between 2002 and 2020.ResultsOne case of TPR-NTRK1 fusion-positive intracranial, extra-axial, high-grade undifferentiated sarcoma (12-year-old boy), one case of LMNA-NTRK1 fusion-positive low-grade infantile fibrosarcoma of the forehead (3-year-old boy), one case of ETV6-NTRK3 fusion-positive inflammatory myofibroblastic tumor (IMT) (3-months-old girl), and six cases of ETV6-NTRK3 fusion-positive infantile fibrosarcoma (median age: 2.6 months, range: 1.6–5.6 months, M: F = 5:1) were reviewed. The Trk immunopositivity patterns were distinct, depending on what fusion genes were present. We observed nuclear positivity in TPR-NTRK1 fusion-positive sarcoma, nuclear membrane positivity in LMNA-NTRK1 fusion-positive sarcoma, and both cytoplasmic and nuclear positivity in ETV6-NTRK3 fusion-positive IMT and infantile fibrosarcomas. Also, the TPR-NTRK1 fusion-positive sarcoma showed robust positivity for CD34/nestin, and also showed high mitotic rate. The LMNA-NTRK1 fusion-positive sarcoma revealed CD34/S100 protein/nestin/CD10 coexpression, and a low mitotic rate. The IMT with ETV6-NTRK3 fusion expressed SMA. Six infantile fibrosarcomas with ETV6-NTRK3 fusion showed variable coexpression of nestin (6/6)/CD10 (4/5)/ S100 protein (3/6).ConclusionsAll cases of NTRK1 and NTRK3 fusion-positive pediatric tumors robustly expressed the Trk protein. A Trk immunopositive pattern and CD34/S100/nestin/CD10/SMA immunohistochemical expression may suggest the presence of NTRK fusion partner genes. LMNA-NTRK1 fusion sarcoma might be a low-grade subtype of infantile fibrosarcoma. Interestingly, more than half of the infantile fibrosarcoma cases were positive for S100 protein and CD10. The follow-up period of TPR-NTRK1 and LMNA-NTRK1 fusion-positive tumors are not enough to predict prognosis. However, ETV6-NTRK3 fusion-positive infantile fibrosarcomas showed an excellent prognosis with no evidence of disease for an average of 11.7 years, after gross total resection of the tumor.

Simultaneous Stripping of Ammonia from Leachate: Experimental Insights and Key Microbial Players

Air stripping is commonly used to remove the ammonia in multistage treatment systems for municipal landfill leachate (LFL). This paper proposes a novel approach combining the process of stripping with biological removal of ammonia, based on simultaneous nitrification and denitrification (SND) in a single hybrid sequencing batch reactor (HSBR). To avoid the accumulation of free ammonia (N-FAN), the shallow aeration system was used for the treatment of raw LFL with N-TAN level of 1520 mg/L and pH 9.24. The mean N-FAN removal efficiency of 69% with the reaction rate of 55 mg L−1 h−1 and mean ammonium (N-NH4+) removal efficiency of 84% with the reaction rate of 44 mg L−1 h−1 were achieved within a month in such an HSBR (R1). The comparative HSBR (R2), with conventional aeration system maintaining the same concentration of dissolved oxygen (DO ≤ 1 mg/L), was removing only trace amounts of N-FAN and 48% of N-NH4+. The quantitative analysis of 16S rRNA genes indicated that the number of total bacteria, Actinobacteria, Bacteroidetes, Firmicutes, and Beta- and Gammaproteobacteria increased during the operation of both HSBRs, but was always higher in R1. Moreover, the bacterial community shift was observed since the beginning of the experiment; the relative abundance of Firmicutes, and Beta- and Gammaproteobacteria increased by 5.01, 3.25 and 9.67% respectively, whilst the abundance of Bacteroidetes and Actinobacteria decreased by 15.59 and 0.95%. All of the surveyed bacteria groups, except Gammaproteobacteria, correlated significantly negatively (p < 0.001) with the concentrations of N-NH4+ in the outflows from R1. The results allow us to suppose that simultaneous stripping and SND in a single reactor could be a promising, cost-effective and easy-to-operate solution for LFL treatment.

Detection of NTRK1/3 Rearrangements in Papillary Thyroid Carcinoma Using Immunohistochemistry, Fluorescent In Situ Hybridization, and Next-Generation Sequencing.

NTRK1/3 rearrangements have been reported in 2.3-3.4% of papillary thyroid carcinoma (PTC) and are regarded as potential therapeutic targets. Recently, the application of immunohistochemistry (IHC) to detect NTRK rearrangements has been widely discussed. The current study aimed to characterize the clinicopathological features of PTC with NTRK1/3 fusions, to examine the utility of pan-TRK IHC, and to compare IHC with fluorescent in situ hybridization (FISH) and next-generation sequencing (NGS). In a cohort of 525 consecutive PTC cases, 60 BRAFV600E-negative cases underwent complete analyses of FISH, and 12 (2.3%) cases with NTRK1/3 break-apart were found. A novel ERC1-NTRK3 fusion was identified by NGS in one case. Pathological features of non-infiltrative tumor border, clear cell change, and reduced nuclear elongation and irregularity were significantly more common in NTRK1/3-rearranged PTC when compared with 48 BRAFV600E-negative non-NTRK1/3 PTC cases. In whole tissue sections, pan-TRK IHC was positive in 3/7 (42.9%) cases with an ETV6-NTRK3 rearrangement including 2 cases with low percentage of stained tumor cells, 2/3 (66.7%) with non-ETV6 NTRK3 rearrangements, and 2/2 (100%) with NTRK1 rearrangements. All FISH-negative cases were negative for pan-TRK in tissue microarray sections. As a result, pan-TRK IHC showed a sensitivity of 58.3% and specificity of 100% for NTRK1/3 rearrangements in BRAFV600E-negative PTC. In conclusion, NTRK1/3-rearranged PTC shared some unique morphologic features. Pan-TRK IHC showed high specificity and moderate sensitivity for NTRK1/3-rearranged PTC and should be interpreted with caution due to staining heterogeneity. Based on the above findings, we propose an algorithm integrating morphology, IHC, and molecular testing to detect NTRK1/3 rearrangements in PTC.

Experimental fatigue behavior of carbon/flax hybrid composites under tensile loading

The aim of the present study is to investigate the mechanical behavior of carbon/flax hybrid composites under static and fatigue tensile loading. The failure characteristics and parameters used in the fatigue tests were deduced from the static ones. The effect of the applied stress level, hybridization and stacking sequences on the stiffness, hysteresis loops, dissipated energy and damping, were studied for a various number of cycles during fatigue tests. The Wohler S-N curves were constructed to investigate the effect of hybridization on the fatigue behavior. The results obtained show that the fatigue performance as well as the fatigue resistance increase with the increase of the volume fraction of carbon fiber. Nevertheless, the damping ratio and the fatigue life increase with the increase of the flax fiber volume fraction.

PREDICTION OF IMPLANTATION RATE IN SINGLE EUPLOID FROZEN-THAWED BLASTOCYST TRANSFER CYCLES BY CULTURE TIME AND/OR RE-EXPANSION RATE AFTER TROPHECTODERM BIOPSY

To examine whether the culture time and/or re-expansion rate after trophectoderm (TE) biopsy can predict implantation rate in single euploid frozen-thawed blastocyst transfer (seFET) cycles. Retrospective cohort study in a private assisted reproductive technology (ART) program. In this retrospective cohort study 397 blastocysts (Day-5 and -6) from 344 patients (35.8 ± 4.2 years of age) were characterized as euploid after preimplantation genetic screening by array comparative genomic hybridization or next generation sequencing. The TE biopsies and the (seFET) cycles were performed at the Kofinas Fertility Group ART clinic from January 2016 to January 2020. All aneuploidy screenings were authorized by patients after consultation. The euploid blastocysts of all participants were divided in two groups: (i) those that were cultured for 1-4 h after TE biopsy (Cultured Group; CG, n=184) and (ii) those that were cryopreserved immediately after TE biopsy (Non-Cultured Group; NCG, n=213). In addition, the CG was further divided in two subgroups based on the re-expansion rate after TE biopsy in relation to the initial blastocoel expansion rate; these subgroups were either: (i) partially re-expanded (pRE) blastocysts (blastocoel formation, but not full re-expansion) or (ii) fully re-expanded, hatching or completely hatched blastocysts (fRE). Implantation rate was assessed in the examined groups and subgroups. No significant difference was detected in the overall implantation rate between the two examined groups, CG and NCG (70% vs 69%, respectively) and between the two subgroups of the CG group, pRE and fRE (70% vs 71%, respectively). In contrast, Day-5 euploid blastocysts that belong to the pRE subgroup demonstrated a significantly higher implantation rate compared to Day-6 euploid blastocysts belonging to the same subgroup (78% vs 52%, respectively, p<0.01). Most importantly, euploid blastocysts cultured for ≤2h after biopsy belonging to the fRE subgroup were characterized by a significantly higher implantation rate when compared with euploid blastocysts cultured for more than 2h after biopsy belonging to the pRE subgroup (83% vs 54% respectively, p<0.05). Implantation rate in seFET cycles can be improved by selecting blastocysts that are able to achieve full re-expansion when cultured for ≤2h after TE biopsy, instead of blastocysts that are cultured for >2h after TE biopsy and they have only partially re-expanded. Further studies are required in order to shed more light on the optimal culture time and/or re-expansion rate after TE biopsy.

Molecular Characterization and Clinical Outcomes in RET-Rearranged NSCLC.

RET rearrangements are an emerging targetable oncogenic fusion driver in NSCLC. However, the natural history of disease and activity of different classes of systemic therapy remain to be defined. Furthermore, molecular testing for RET is not yet routine, and the optimal method of testing is unclear. We present a comparative analysis of molecular profiling with fluorescence in situ hybridization (FISH) or next-generation sequencing (NGS) and treatment outcomes. This study was a retrospective analysis of patients treated at the National Cancer Centre Singapore. Baseline demographics and treatment outcomes were collected. A total of 64 patients were included, with a median age of 62 years (range: 25-85), 56% were women, 77% were of Chinese ethnicity, 95% had adenocarcinoma, and 69% were never smokers. RET rearrangement was detected by FISH in 30 of 34 patients (88%), NGS in 40 of 43 patients (93%), and with discordant results in seven of 13 patients (54%) tested with both methods. Of 61 patients with stage IIIB/IV or recurrent disease, prevalence of central nervous system metastases was 31% and 92% received palliative systemic therapy. Overall survival was prolonged in patients treated with a selective RET tyrosine kinase inhibitor versus untreated patients (median 49.3 versus 15.3 mo; hazard ratio [HR]: 0.16, 95% confidence interval [CI]: 0.06-0.40, p < 0.001). However, it was not different in patients treated with immunotherapy versus untreated patients (median 37.7 versus 49.3 mo; HR: 1.30, 95% CI: 0.53-3.19, p= 0.53). Overall survival was also prolonged in patients with CCDC6-RET fusion versus those with KIF5B-RET fusion (median 113.5 versus 37.7 mo; HR: 0.12, 95% CI: 0.04-0.38, p= 0.009). In RET-rearranged NSCLC, selective RET tyrosine kinase inhibitor therapy is associated with improved survival outcomes, especially in patients with CCDC6-RET fusion. However, immunotherapy has poor efficacy. NGS and FISH testing methods may also result in substantial discordance.

Hybrid transcriptome sequencing approach improved assembly and gene annotation in Cynara cardunculus (L.)

BackgroundThe investigation of transcriptome profiles using short reads in non-model organisms, which lack of well-annotated genomes, is limited by partial gene reconstruction and isoform detection. In contrast, long-reads sequencing techniques revealed their potential to generate complete transcript assemblies even when a reference genome is lacking. Cynara cardunculus var. altilis (DC) (cultivated cardoon) is a perennial hardy crop adapted to dry environments with many industrial and nutraceutical applications due to the richness of secondary metabolites mostly produced in flower heads. The investigation of this species benefited from the recent release of a draft genome, but the transcriptome profile during the capitula formation still remains unexplored. In the present study we show a transcriptome analysis of vegetative and inflorescence organs of cultivated cardoon through a novel hybrid RNA-seq assembly approach utilizing both long and short RNA-seq reads.ResultsThe inclusion of a single Nanopore flow-cell output in a hybrid sequencing approach determined an increase of 15% complete assembled genes and 18% transcript isoforms respect to short reads alone. Among 25,463 assembled unigenes, we identified 578 new genes and updated 13,039 gene models, 11,169 of which were alternatively spliced isoforms. During capitulum development, 3424 genes were differentially expressed and approximately two-thirds were identified as transcription factors including bHLH, MYB, NAC, C2H2 and MADS-box which were highly expressed especially after capitulum opening. We also show the expression dynamics of key genes involved in the production of valuable secondary metabolites of which capitulum is rich such as phenylpropanoids, flavonoids and sesquiterpene lactones. Most of their biosynthetic genes were strongly transcribed in the flower heads with alternative isoforms exhibiting differentially expression levels across the tissues.ConclusionsThis novel hybrid sequencing approach allowed to improve the transcriptome assembly, to update more than half of annotated genes and to identify many novel genes and different alternatively spliced isoforms. This study provides new insights on the flowering cycle in an Asteraceae plant, a valuable resource for plant biology and breeding in Cynara and an effective method for improving gene annotation.

Prevalence of Aminoglycoside Resistance Genes and Molecular Characterization of a Novel Gene, aac(3)-IIg, among Clinical Isolates of the Enterobacter cloacae Complex from a Chinese Teaching Hospital.

Members of the Enterobacter cloacae complex are important opportunistic human pathogens capable of causing a wide variety of infections. During recent decades, aminoglycoside-resistant E. cloacae complex isolates have increasingly been reported and have become a major concern. Here, we employed high-throughput sequencing in combination with specific PCR assays to investigate the prevalence of aminoglycoside resistance genes among 170 isolates of the E. cloacae complex collected from a teaching hospital in Wenzhou, China. A total of 12 known genes [aphA-1, strA, strB, aac(6')-IIc, aadA2, aac(3)-IId, aadB, aadA1, rmtB, armA, aadA5, and aac(6')-Ie-aph(2'')-Ia] and 1 novel gene [aac(3)-IIg] were identified, with aphA-1 (71.18%), strA (55.29%), and strB (52.35%) being the most prevalent, and aac(3)-IIg was detected with a positive rate of 21.76% (37/170). The aac(3)-IIg gene was 810 bp in length and encoded a protein that shared 72 to 78% identities with previously known AAC(3)-II aminoglycoside 3-N-acetyltransferases. The MICs of gentamicin and tobramycin were 512 μg/ml and 64 μg/ml, respectively, when aac(3)-IIg was cloned into Escherichia coli DH5α. All aac(3)-IIg-positive isolates exerted broad aminoglycoside resistance profiles, mediated by the coexistence of multiple resistance genes. Moreover, aminoglycoside resistance and resistance genes were found to be transferable in most strains (24/37). Nevertheless, pulsed-field gel electrophoresis (PFGE) and dendrogram analysis showed clonal diversity among these isolates. S1 nuclease PFGE, Southern hybridization, and whole-genome sequencing indicated that aac(3)-IIg was located on transferable as well as nontransferable plasmids of various sizes. The analysis of the genetic environment suggested that aac(3)-IIg is embedded within a class 1 integron, with IS26 playing an important role in its mobility.

Abstract 4254: Quality assessment platform for molecular pathology from non small cell lung cancer histopathology images using deep learning: A nationwide study of central laboratory in South Korea

Abstract Molecular testing became the most important part of lung cancer diagnostics. Integration of various molecular testing such as real-time PCR, fluorescent in situ hybridization (FISH) and next-generation sequencing into routine clinical practice and their systemic quality control is challenging to a pathologist and even more in the central laboratory. In this study, we trained a variant of DenseNet, a convolutional neural network model with the highest performance in image classification and suitable for quality assessment. Train dataset is composed of the arbitrarily classified dataset made and assessed by a professional pathologist during routine molecular test using whole-slide tissue images and molecular profiles including DNA density and the signal intensity of FISH obtained from 15,000 tests from lung cancer biopsies and resections, which were sent to the central laboratory for molecular studies from 45 hospitals nationwide. The performance of our platform is tested and compared to the quality assessment dataset made from each a general pathologist and a technician. The performance of our method for classifying DNA concentration and the signal intensity of FISH is comparable to that of a general pathologist and we are under improving its average area under the curve (AUC) over 0.95. This result suggests that deep learning model based on a convolutional neural network can assist pathologists for rigorous assessing quality of tissue morphology and signal of FISH for tissue samples with various molecular quality before or during the molecular test and, furthermore, can be applied to quality control of lung cancer molecular testing in a central laboratory. Citation Format: Tae-Jung Kim, Ho Chul Kang, Yonggeun Lee, Seokman Seo, Donghwan Kim. Quality assessment platform for molecular pathology from non small cell lung cancer histopathology images using deep learning: A nationwide study of central laboratory in South Korea [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4254.

Gene Fusions in Ocular Adnexal Tumors

To highlight the increasing importance of gene fusions in the diagnosis, prognosis, and therapy of ocular adnexal tumors. Perspective. A focused review of gene fusions, their pathogenic mechanism, and gene fusion detection methods in lacrimal gland and primary orbital and ocular adnexal soft tissue tumors; reappraisal of diagnostic, prognostic, and therapeutic approach to ocular adnexal tumors in light of emerging molecular genetic data. The widespread implementation of fluorescence insitu hybridization and next-generation sequencing methods in pathology practice has led to identification of recurrent gene rearrangements and fusions in a variety of tumors. As a result, molecular genetic methods have become the gold standard for diagnosis of tumors with overlapping histology and immunophenotype, such as small round blue cell tumors. Identification of canonic gene fusions has led to development of sensitive and specific immunohistochemical markers, such as STAT6 in solitary fibrous tumor. In addition to diagnostic accuracy, gene fusions have prognostic implications, such as unfavorable prognosis of PAX3-FOXO1 fusion in alveolar rhabdomyosarcoma. Finally, recognition of gene fusions as a driving mechanism in neoplasia has led to development of U.S. Food and Drug Administration-approved targeted therapies, such as TRK inhibitors for NTRK fusion-positive cancers. The discovery of recurrent gene fusions in various tumors, including those involving ocular adnexa, has led to a deeper insight into the molecular mechanisms of these neoplasms, revolutionizing our approach to their diagnosis, prognostication, and therapy.